[unreadable] Nerve growth factor (NGF) and other neurotrophic factors (NTFs) are produced and released in target tissues to activate specific receptors on the distal axons of innervating neurons. The signals thus produced are retrogradely transported to the cell bodies to regulate cytosolic and nuclear events important for survival and differentiation. An important unresolved issue is the mechanism(s) by which signals generated by NGF in axon terminals are transmitted. In the last funding period, we tested the signaling endosome hypothesis which states that the NGF signal is retrogradely transported through endosomes that contain NGF bound to its activated TrkA receptor in complex with associated signaling proteins. We gathered support for the "signaling endosome" hypothesis, and isolated such organelles, but many questions remain as to the structure and function of signaling endosomes. The hypothesis tested in this application is that signaling endosomes are an important source of retrogradely transmitted NGF signals. In the proposed Aims, we will examine the signaling pathways that use endosomes to signal, characterize the proteins that participate, examine signaling from endosomes in vitro and in vivo, and test the role that endosome-derived signals play in maintaining mature DRG neurons, cells that respond robustly to NGF but that are independent of NGF for survival. In Aim 1, examining intact cells and endosome-containing fractions, we will define signaling pathways that employ endosomes. Immunostaining and confocal microscopy will be used as will biochemical studies of signaling proteins. In Aim 2, using compartmented cultures, we will test what signaling pathways use endosomes to retrogradely transport NGF signals from axon terminals to cell bodies. We will define the cell biological steps required to make and traffic signaling endosomes. Endocytosis of NGF will be inhibited using bead-bound NGF. Other steps critical to endocytosis and trafficking events will be inhibited using Lentivirus to express dominant negative isoforms of proteins or to induce RNAi to suppress synthesis of endogenous proteins. In Aim 3, we will explore the cellular events induced by retrogradely transported signaling endosomes at the level of downstream signaling events, gene expression and cellular morphology. Finally, in Aim 4, we will examine the biochemical properties of signaling endosomes isolated by FACS sorting or by magnetic immuno-adsorption and will test signaling from these endosomes in vitro and in vivo. The proposed studies are expected to provide new insights into the cell biology of NGF actions and may help to elucidate a role for failed signaling through endosomes in the pathogenesis of Alzheimer's disease and Down syndrome. [unreadable] [unreadable]